Excitation-contraction coupling in the heart confers a tight linkage between the L-type calcium channel and the intracellular environment so that Ca2+ ingress upon depolarization triggers contraction. We have been studying the complex architecture, regulation and receptor drug sites on the subunits that comprise the cardiac calcium channel. Considering the importance of this channel in maintaining normal cardiac function, it is not surprising that defects have been found in some types of human heart failure. This continuing project focuses on subunit functional interaction in vivo on the normal and falling heart. The latter is produced in the mouse by a specific increase of L-type calcium channel subunits in a transgenic mouse, which has provided us with a cardiomyopathy very close to the human type. Transgenic "remodeling" of the heart is not only a convenient method of altering subunit stoichiometry in vivo, but provides a way to approach mechanisms of heart failure in a logical manner. The long-term objective is characterization of calcium channel regulation, in terms of the pore, subunit importance, and calcium antagonist receptor functions. The added feature in this renewal application is the transgenic and knockout approach, encompassing coordinated physiological, biochemical and microanatomical methodology. We hope to provide further molecular information on the normal L-type cardiac channel and its possible role in the diseased heart. The specific aims are: 1) to over-express the human cardiac alpha1 subunit specifically and only in the myocytes of transgenic mice. Physiological function, genotype frequency, and expression levels of the transgene, as well as comprehensive cardiac pathological assessment, hopefully will yield important new information. 2) To eliminate the functional effects of the beta-subunit in mouse heart by generating a dominant negative expression system. The high affinity alpha1 interactive domain (AID) will be over- expressed in a cardiac-specific manner, and this will then act as a trap for beta subunits. Characterization is as in Aim 1. We hope to provide new information n the role of the beta-subunit in vivo. 3) To generate conditional knockout mice lacking the alpha2/delta subunit in the heart. Studying these mice, again on the molecular, cellular and whole organ level, should give us valuable information on the role of this subunit in vivo. 4) To continue studies on the pore-lining region of calcium channels. We suggest that these studies will give us specific information concerning some structural features of the pore that are involved in regulation of the channel.